The secretion of gonadotropin is controlled by multiple signals. GnRH is a primary regulator and its input is necessary for expression of each of the gonadotropin genes. Estrogen (E2) from the ovary feedbacks to both the hypothalamus and pituitary to positively and negatively regulate gonadotropes. It was demonstrated that E2 suppresses basal activity and enhances GnRH responsiveness of alpha-promoter, which gives rise to the sexual dimorphism in the degree of GnRH stimulation of alpha-promoter. Our preliminary data and other studies suggest that transcription factor CREB may play a role in E2 regulation of alpha-promoter activity. The goal of this project is to elucidate the molecular mechanism by which E2 alters the function of gonadotrope cells. Aim 1 is to examine whether E2 induces the phosphorylation of CREB in the pituitary. Both in vitro and in vivo models will be used and the CREB phosphorylation will be tested with an antibody that differentiates between CREB and phosphorylated CREB. In addition, transfection experiments with a CREB dominant negative form will be performed to establish that the phosphorylation of CREB will cause the increase in alpha-promoter responsibility to GnRH. Aim 2 is to identify proteins that might be involved in increasing CREB phosphorylation i the pituitary with PCR-select cDNA subtraction. Transfection assay will be performed to confirm the role off regulation of a-promoter activity. This study will provide new insights into molecular basis of E2 regulation of gonadotropes and provide a platform for rational controlling and manipulating gonadotrope dysfunction.